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Aerobic degradation of 2,4,6-TCP content in ECF bleached effluent
Environment International 29 (2003) 459 – 465 www.elsevier.com/locate/envint
Aerobic degradation of 2,4,6-TCP content in ECF bleached effluent J. Correa a, V.M. Domı´nguez b, M. Martı´nez a, G. Vidal b,* a
Department of Microbiology, Biological Sciences Faculty, University of Concepcio´n, P.O. Box 160-C, Concepcion, Chile b Environmental Science Center EULA-Chile, University of Concepcio´n, P.O. Box 160-C, Concepcion, Chile Received 4 September 2002; accepted 12 December 2002
Abstract Elemental chlorine-free (ECF) bleach effluents from kraft mill are characterised by: a chemical organic demand/biological organic demand (COD/BOD5) ratio of 4, chlorophenol content with low chlorine substitution, and toxicity. The effect of increasing the concentration of 2,4,6-trichlorophenol (2,4,6-TCP) content in ECF bleaching sequence effluent on the degradative activity of bacterial communities present in an aerobic system treatment was studied. An aerobic lagoon (AL) was used as a typical secondary treatment of kraft-mill effluent. AL displays a high performance of BOD5 degradation (up to 90%); however, only 40% of the COD was removed. Simultaneously, the AL system shows a high ability to biodegrade 2,4,6-TCP up to 237 mg/l day. Kinetic parameters of the 2,4,6-TCP biodegradation by aerobic bacteria were determined. The Ks and Ki values were 34.3 and 50 mg/l 2,4,6-TCP, respectively. Moreover, the tolerance of aerobic bacteria was observed up to 1.3 g/l 2,4,6-TCP. D 2003 Elsevier Science Ltd. All rights reserved. Keywords: 2,4,6-TCP; Aerobic biodegradation; Isolation strain; ECF effluent
1. Introduction The pulp-and-paper industry has made substantial changes to pulping and bleaching technologies in order to minimise the formation and discharge of chlorinated organic material into aquatic environments (Videla and Diez, 1997). In the bleach plant, the major change has been the replacement of elemental chlorine dioxide, resulting in elemental chlorine-free (ECF) bleach effluents with lower absorbable organic halogens (AOX) and lower toxicity (Stauber et al., 1996). The biological demand for oxygen (BOD5) is reduced in the secondary treatment; however, specific compound removal (such as chlorophenols) is not always monitored. Diverse chlorolignin derivates of both low molecular weight (such as chlorophenols, chlorocatecols, chloroguaiacols) and high molecular weight are generated when ECF technologies are implemented (Lage et al., 1999; Vidal et al., 2001). In particular, 2,4,6-trichlorophenols (2,4,6-TCPs) are found as a major component of ECF bleach effluents (Andreoni et al., 1998; Diez et al., 2002). The study of the
* Corresponding author. Tel.: +56-41-204002; fax: +56-41-242546. E-mail address: [email protected] (G. Vidal).
role of microorganisms in chloroorganic removal during the aerobic treatment of bleached kraft-mill effluents (BKMEs) has been carried out recently (Fulhorpe et al., 1993). Studies show that chlorinated phenolics removal can be mineralized by aerobic bacteria in consortium (Vidal et al., 1997; Graves et al., 1995; Hall and Randle, 1994), or in a pure culture (Valenzuela et al., 1997; Domı´nguez et al., 2002). However, there are no specific studies that show evidence of the biodegradation of 2,4,6-TCP in effluents generated by the ECF process when Pinus radiata is used as the raw material. In order to predict the behaviour of these compounds, it is important to consider the kinetic biodegradation in the aerobic system. The Andrews kinetics (Eq. (1)) was used to describe the inhibition of 2,4,6-TCP and to evaluate the inhibition constant (Gu and Korus, 1995), considering the initial rate of the substrate degradation (rs) for each concentration evaluated (Vidal et al., 1997): rs ¼
dS X lm S ¼ dt YX =S Ks þ S þ SK2 i
ð1Þ
where S is the substrate concentration, X is the cell concentration, Ks is the Monod half saturation constant, Ki is an inhibition constant, lm is the maximum growth rate, and YX/S is the cell yield.
0160-4120/03/$ - see front matter D 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0160-4120(03)00002-3
460
J. Correa et al. / Environment International 29 (2003) 459–465
The aim of this work was to investigate the effect of increasing the concentration of 2,4,6-TCP content in ECF bleaching sequence effluent on the degradative activity of bacterial communities present in an aerobic treatment system.
2. Materials and methods 2.1. Wastewater The ECF effluent was obtained from a kraft mill described by Vidal et al. (2001). The effluent was supplemented with (NH4)2SO4 and KH2PO4 as a nitrogen and phosphate source (BOD5:N:P = 100:5:1). A stock solution of 2000 mg/l 2,4,6-TCP (Sigma, St. Louis, MO, USA) neutralized with NaOH solution (0.1 N) was prepared. Different 2,4,6-TCP concentrations were added to the ECF effluent. Table 1 shows the main physical – chemical characteristics of this effluent.
2.4. Kinetics of 2,4,6-TCP biodegradation The kinetics of 2,4,6-TCP biodegradation was measured using a batch system. Erlenmeyer flasks (250 ml) were incubated with biomass from the AL. Concentrations of 5, 10, 20, 40, and 80 mg/l 2,4,6-TCP were inoculated according to Domı´nguez et al. (2002). All of the assays were carried out in triplicate. The flasks were incubated in a shaker at 150 rpm, in the dark, at 25 F 2 jC. The variation of the 2,4,6-TCP concentration and cell concentration was analyzed according to the procedure described in Analytical Methods, during a period of 5 days, until total mineralization. The 2,4,6-TCP concentration was determined in the liquid phase for each assay by spectrophometry according to the process described by Aranda et al. (1999). The sample extraction was carried out according to the instructions of Veith and Kiwus (1997). The complete mineralization of the 2,4,6-TCP was confirmed by high-performance liquid chromatography (HPLC). 2.5. Isolation strains and bacterial viability
2.2. Inoculum A consortium of aerobic bacteria [4.1 g/l volatile suspended solid (VSS) and 5.9 g/l total suspended solid (TSS)] was inoculated in an aerobic lagoon (AL). This consortium arises from an activated sludge system that treats bleached kraft-mill effluent. 2.3. Continuous biodegradation system An AL (3.4 l) was used for biological effluent treatment. As is indicated in Fig. 1, an AL consists of an aerated zone and a settling zone. Oxygen concentration was maintained above 2 mg/l by a diffuser system. The AL operation was divided in two phases. In phase 1, the system was fed by means of a peristaltic pump with only ECF effluent; the hydraulic retention time (HRT) and organic load rate (OLR) in the system were 2 days and 184 mg/l day BOD5, respectively. In phase 2, the system was fed with ECF effluent supplemented with increasing 2,4,6-TCP concentrations; the OLR and HRT were 69 mg/l day BOD5 and 5 days, respectively. The toxic load rate (TLR) was maintained between 5.5 and 237 mg/l day 2,4,6-TCP.
Table 1 Physical – chemical characteristics of ECF effluent Parameter
Range
Averagea
pH COD (g/l) BOD5 (g/l) Total phenolic compounds UV215 (mg/l) Phenols (mg/l) Tannin and lignin (mg/l) TSS (g/l)
3.5 – 10.6 0.8 – 1.9 0.2 – 0.5 190.0 – 350.0 0.9 – 1.2 44.0 – 64.0 1.2 – 3.1
7.3 1.2 0.3 321.7 1.1 52.2 2.2
a
Average values correspond to 11 determinations.
Bacterial viability was determined in R2A agar plates and incubated at 25 jC for 5 days (Herbert, 1990; Godoy et al., 1999; Martı´nez et al., 1999). All experiments were carried out in triplicate. Samples obtained after 3 days of incubation were placed on R2A agar plates and incubated for 48 h at 25 jC in order to isolate bacteria strains that are present in ALs and that have the ability to degrade 2,4,6-TCP. Bacterial colonies with different morphological characteristics were first selected and frequently checked for purity in R2A agar plates and then characterised for Gram-strain type. Gramnegative bacilli were categorised by oxidative or fermentative capabilities in glucose H2S medium (Ward et al., 1986). The isolated cells were incubated in Erlenmeyer flasks (250 ml) at 25 jC with constant shaking (150 rpm) using the same method described by Godoy et al. (1999). Minimum medium (MM) was added to each of the seven Erlenmeyer flasks (containing 5, 10, 20, 40, 80, 160, and 360 mg/l 2,4,6TCP, respectively)—with the MM, effluent, and 2,4,6-TCP being the sole carbon and energy source. Additionally, we investigated the biodegradation of 2,4,6-TCP using R2A broth in order to evaluate the behavior of bacterial strain growth (Domı´nguez et al., 2002). The 2,4,6-TCP biodegradation and bacterial growth samples from each Erlenmeyer flask were analyzed daily for 7 days. 2.6. Analytical methods VSS, TSS, chemical organic demand (COD), biological organic demand (BOD5), phenols, and total phenolic compounds (UV215) were measured according to standard methods (APHA-AWWA-WPCF, 1985). Samples for determining the concentration of COD, BOD5, and UV215 were membrane-filtered (0.45 Am). The concentration of UV215
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Fig. 1. Aerobic lagoon: (1) influent, (2) pump, (3) diffusers, (4) aeration zone, (5) settling zone, (6) final effluent.
was determined in a 1-cm quartz cuvette by diluting the samples to less than 0.8 absorbance units (0.025 M borate was used as a buffer providing a pH of 9.1).
3. Results and discussion 3.1. Aerobic treatment Fig. 2 and Table 2 show the AL performance during 147 days of operation in which the TLR ranged from 5.5 to 237 mg/l day 2,4,6-TCP (Fig. 2a). The reactor was kept stable during the whole operation, maintaining the pH of the effluent between 7 and 8. In the first phase (0 –25 days of operation), when only ECF effluents were fed, almost 40% wt/wt of the COD was removed by the aerobic system (Fig. 2b). In contrast, BOD5 removal was between 90% and 96% wt/wt, whereas tannin and lignin removal was only around 13 –45% wt/wt. However, total phenolic compounds, measured as UV215, were poorly removed (8– 25% wt/wt), which indicates only a minor removal of these kinds of compounds. The influent COD/BOD5 ratio was 4, indicating that kraft-mill effluent is only partially biodegradable. Similar results were shown by Diez et al. (2002). Moreover, they found that the effluent biodegradation is highly influenced by the HRT in the activated sludge system; optimal HRT in this case was 48 h. In the second phase of the AL operation, ECF effluent was supplemented with increasing concentrations of 2,4,6TCP. The aerobic bacteria consortium displayed the ability to biodegrade the 2,4,6-TCP. However, during the operation between 26 and 36 days (TLR of 4 mg/l day 2,4,6-TCP), the aerobic system showed an accumulation of 2,4,6-TCP. Fig. 3a shows the profile of the 2,4,6-TCP content in the AL effluent; absorbance at 285 nm showed that the accumulation of the chlorophenols occurred between 26 and 34 days of operation, after which the 2,4,6-TCP was degraded. This phenomenon was also observed in each case when the TLR of 2,4,6-TCP was increased. Fig. 3b shows the 2,4,6-
TCP behaviour in the AL effluent, between 79 and 89 days of operation (TLR of 44 mg/l day 2,4,6-TCP). These results suggest that aerobic bacteria can degrade 2,4,6-TCP in the presence of ECF bleached effluent with high molecular weight (Vidal et al., 2001) and content of COD (mainly chloroaromatic containing), but need an adaptation period at a high concentration of the toxic. After that period, removal efficiency of 2,4,6-TCP during the process was always above 95%. Graves et al. (1995) also observed removal of chlorophenols from ECF effluent. They found AOX removal ranging from 15% to 43%, while removal of the individual chlorinate species varied from 12% to 100%. Hence, the concentration and viability of the microorganism population are important in the performance of the AL. In our experiments, the microorganism concentration increased from 2.38 to 3.37 g/l SSV and the ratio SSV/SST was between 0.76 and 0.68. Under these conditions, the 2,4,6TCP did not affect the metabolic activity of the microorganisms when the concentration of the toxic was increased from 20 to 1300 mg/l 2,4,6-TCP. Apart from the bacteria identifications in the system, we also observed the presence of a significant protozoan population in the AL, which reinforced the ability of the aerobic system to degrade the 2,4,6-TCP. 3.2. Kinetics of 2,4,6-TCP biodegradation In order to determine the kinetic parameters of the aerobic bacteria, we evaluated in batch system the bacterial capability to degrade 5, 20, 40, and 80 mg/l 2,4,6-TCP until mineralization. 2,4,6-TCP concentration was supplied with ECF effluents as the sole carbon and energy source in these assays. Assays with initial concentrations of 2,4,6-TCP between 20 (Fig. 4b) and 40 mg/l (Fig. 4c) showed a complete degradation of the chlorophenol at 120 h. After this, biomass could grow to 1.0 109 cfu/ml. The increase in biomass after the complete degradation of 2,4,6-TCP has been described as an intracellular dynamics that is often associated with phenol metabolism (Kim and Hao, 1999).
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Fig. 2. Performance of AL. (a) TLR (.). (b) COD (o) and 2,4,6-TCP removal (.). (c) Cell growth log (n).
A different behaviour was displayed in assays with 80 mg/l 2,4,6-TCP (Fig. 4d). A lag phase of about 50 h was observed. This indicates the toxic effect of the chloropheTable 2 Performance of aerobic lagoon during the operation Period (days)
Influent TLR (mg/l day 2,4,6-TCP)
2,4,6-TCP (mg/l)
2,4,6-TCP (% wt/wt)
0 – 25 26 – 55 56 – 75 76 – 86 89 – 107 110 – 133 134 – 147
0 5.5 12 44 72 114 237
0 30 70 250 400 650 1300
0 76.1 87.5 98.3 96.3 99.1 97
TLR = toxic load rate.
Effluent
nols and its inhibitory effect on the specific enzymatic activities in a batch system. Similar results were observed by Valenzuela et al. (1997). In our assays, no biodegradation was observed until 50 h. Also, from 24 to 50 h, the population increased to 1.0 104 cfu/ml. However, adaptation was observed and 80 mg/l 2,4,6-TCP was completely degraded at 120 h. The maximum degradation rate (1.1 mg/l h 2,4,6-TCP) and cell density (1.0 107 cfu/ml) were measured in the period higher than 120 h. The Andrews model was considered in order to obtain the main kinetic constant (saturation and inhibitory constant) of this aerobic bacteria in the presence of 2,4,6-TCP. Model parameters were determined by minimising the deviations between experimental data and model predictions of the degradation rate; the r2 value was 0.99. Thus, 34.3 and 50 mg/l were determined as saturation (Ks) and inhib-
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Fig. 5. Initial degradation rate of 2,4,6-TCP. Experimental data (n) and prediction by the Andrews model (—).
Fig. 3. Behaviour of 2,4,6-TCP in continuous system at TLR of (a) 5.5 mg/l day and (b) 44 mg/l day.
ition (Ki) constants, respectively, using Table Curve software. Fig. 5 shows the model fitted to the experimental data. The different model parameters for the degradation of 2,4,6-TCP (Table 3) can be explained as the difference in substrate transport across the membranes of the different cells. Little is known about how aromatic compounds and other hydrophobic compounds enter cells—although diffusion is the most cited mechanism (Reardon et al., 2000). Table 3 shows values of Ks and Ki similar to those obtained by Gu and Korus (1995) and Vidal et al. (1997).
Fig. 4. Degradation of 2,4,6-TCP with MM (.) and cell growth (o). Initial concentration of 2,4,6-TCP in each assay: (a) 5 mg/l, (b) 20 mg/l, (c) 40 mg/l, and (d) 80 mg/l.
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Table 3 Kinetic constants determined by the Andrews model in the degradation of 2,4,6-TCP Cells source Aerobic consortium from aerated lagoon-treated pulp kraft-mill effluent (raw material: pine) Isolated strain from river sediment Aerobic consortium from aerated lagoon-treated pulp kraft-mill effluent (raw material: eucalyptus) Flavobacterium sp. ATCC 39723 Consortium of six isolated and identified strains
Ks (mg/l)
Ki (mg/l)
Reference
34.3
50
This works
107 48.1
14 n.d.
156 n.d.
of COD (average 1.2 g/l COD) and toxic compounds. As a result, the bacteria needed to simultaneously develop both the ability to degrade nontoxic organic matter and to degrade toxic compounds. The study of the synergism phenomena between bacteria and the mechanism of the 2,4,6-TCP biodegradation in the presence of a co-substrate will be the objective for future works.
Domı´nguez et al. (2002) Vidal et al. (1997)
21
Gu and Korus (1995)
610
Kharoune et al. (2002)
n.d. = not determined.
Moreover, the values obtained in this work are very similar to those obtained by Vidal et al. (1997), who studied bacteria indigenous to Spain rather than Chile. However, in both cases, the substrates were ECF effluents and an aerobic consortium from AL-treated kraft-mill effluent. The value of the Ki constant (50 mg/l 2,4,6-TCP) helps to explain the performance of the continuous AL system. That means that aerobic biomass can be inhibited at concentrations below or similar to 50 mg/l 2,4,6-TCP; however, adaptation of the aerobic bacteria can improve the system performance. 3.3. Isolation strains Six strains were isolated in order to study the ability to degrade 2,4,6-TCP. Unfortunately, we did not observe evidence of the ability of the strains to degrade the toxic compounds. However, tolerance to 2,4,6-TCP was noted in an isolated condition, inoculated in R2A broth or saline solution (MM) with 2,4,6-TCP. Interestingly, degradative activity was detected only in the presence of cellulose effluent or in R2A broth inoculated with effluent (data not shown). Both conditions include different kinds of aerobic bacteria. The bacteria strains, in isolated condition, were unable to degrade 2,4,6-TCP when used as the sole carbon source or added to R2A broth, thus suggesting that degradative activity is a consequence of a bacterial consortium activity (Kharoune et al., 2002). Another possibility is that bacteria with degradative properties are found in viable but not cultivable conditions (Kejelleberg et al., 1987). In previous studies, we have isolated two different strains (Shingofixis chilensis S37, Ralstomia sp.) from river sediment with the ability to degrade 2,4,6-TCP (Domı´nguez et al., 2002; Godoy et al., 1999, 2002; Martı´nez et al., 1999). In both cases, strains were in contact with a low content of organic matter (below 10 mg/l COD). In contrast to these studies, we exposed aerobic bacteria to high concentrations
4. Conclusions The continuous AL system shows a BOD5 removal between 90% and 96% wt/wt. However, only 40% of the COD was removed. Partial biodegradation can be forecast by the COD/BOD5 relationship (ratio of 4) of the kraft-mill effluent. At the same time, the AL system shows a high ability to biodegrade 2,4,6-TCP up to 237 mg/l day 2,4,6TCP. Tolerance of aerobic bacteria was observed up to 1.3 g/ l 2,4,6-TCP in continuous system. However, the kinetics of the aerobic consortium determined in batch conditions shows Ks and Ki values of 34.3 and 50 mg/l 2,4,6-TCP, respectively. Acknowledgements This work was supported by project FONDECYT 1010644 and grants 200.036.020-1.0 and 200.091.028-1.0 of the Direccio´n de Investigacio´n of the Universidad de Concepcio´n, Chile. References Andreoni V, Baggi G, Colombo M, Cavalca L, Zangrossi M, Bernasconi S. Degradation of 2,4,6-trichlorophenol by a specialized organism and by indigenous soil microflora: bioaugmentation and self-remediability for soil restoration. Lett Appl Microbiol 1998;27:86 – 92. American Public Health Association (APHA), American Water Works Association (AWWA), Water Pollution Control Federation (WPCF). Standard methods for examination of water and wastewater. 16th ed. 1985 [Washington]. Aranda C, Godoy F, Gonza´lez B, Homo J, Martı´nez M. Effects of glucose and phenylalanine upon 2,4,6-trichlorophenol degradation by Pseudomonas paucimobilis S37 cells in a no-growth state. Microbios 1999; 100:73 – 82. Diez MC, Castillo G, Aguilar L, Vidal G, Mora ML. Operational factors and nutrients effect on activated sludge treatment for phenolic compounds degradation from Pinus radiata kraft-mill effluent. Bioresour Technol 2002;83(2):131 – 8. Domı´nguez V, Correa J, Vidal G, Lo´pez A, Martı´nez M. 2,4,6-Trichlorophenol degradation by river sediment exposed to bleached kraft-mill discharge. Environ Contam Toxicol B 2002;69(4):463 – 70. Fulhorpe RA, Liss S, Allen G. Characterization of bacteria isolated from a bleached kraft pulp mill wastewater treatment system. Can J Microbiol 1993;39:13 – 24. Godoy F, Zenteno P, Cerda F, Gonza´lez B, Martı´nez M. Tolerance to trichlorophenols in microorganism from polluted and pristine site of a river. Chemosphere 1999;38:655 – 62. Godoy F, Vancanney M, Martı´nez M, Steinbu¨chel A, Swings J, Rehm BHA. Shingopixis chilensis sp. Nov., a chlorophenol degrading bacte-
J. Correa et al. / Environment International 29 (2003) 459–465 rium which accumulates polyhydroalkanoates. Int J Syst Evol Microbiol 2002 [in press]. Graves J, Jameel H, Joyce T. Removal of chlorophenolics from an aerated lagoon. Tappi J 1995;78(5):99 – 104. Gu Y, Korus RA. Effects of p-Cresol and chlorophenols on pentachlorophenol biodegradation. Biotechnol Bioeng 1995;47:470 – 5. Hall ER, Randle WG. Chlorinated phenolics removal from bleached kraft mill wastewater in three secondary treatment processes. Water Sci Technol 1994;29:177 – 87. Herbert RA. Methods for enumerating microorganisms and determining biomass in natural environments. In: Grigorova R, Norris JR, editors. Methods in microbiology, vol. 22. London: Academic Press; 1990. p. 627. Kejelleberg S, Hermansson M, Marden P, Jones GW. The transient phase between growth and nongrowth of heterotrophic bacteria, with emphasis on the marine environment. Annu Rev Microbiol 1987;41:25 – 49. Kharoune L, Kharoune M, Lebeault JM. Aerobic degradation of 2,4,6trichlorophenol by a microbial consortium—selection and characterization of microbial consortium. Appl Microbiol Biotechnol 2002;59(1): 112 – 7. Kim MH, Hao OJ. Cometabolic degradation of chlorophenols by Acinetobacter species. Water Res 1999;33:562 – 74. Lage LEC, Sant’Anna Jr GL, Nobrega R. Molecular weight distribution of chlorolignin in bleached kraft effluent by GPC and ultrafiltration. Bioresour Technol 1999;68:63 – 70.
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Martı´nez M, Campos A, Garcı´a A, Gonza´lez CL. Marine bacteria tolerant to chlorophenols. Bull Environ Contam Toxicol 1999;62:272 – 7. Reardon KF, Mosteller DC, Bull Rogers JD. Biodegradation kinetics of benzene, toluene, phenol as single and mixed substrates for Pseudomonas putida F1. Biotechnol Bioeng 2000;69:385 – 400. Stauber J, Gunthorpe L, Woodworth J, Munday B, Krassoi R, Simon J. Comparative toxicity of effluent from ECF and TCF bleaching of eucalypt kraft pulps. Tappi J 1996;49(3):184 – 8. Valenzuela J, Bumann U, Ce´spedes R, Padilla L, Gonza´lez B. Degradation of chlorophenols by Alcaligenes eutrophus JMP134 (pJP4) in bleached kraft mill effluent. Appl Environ Microbiol 1997;63(1):227 – 32. Veith GD, Kiwus LM. An exhaustive steam-distillation and solvent extraction unit for pesticides and industrial chemical. Bull Environ Contam Toxicol 1997;17:631 – 6. Vidal G, Kennes C, Me´ndez R, Lema JM. Aerobic degradation of 2,4,6trichlorophenol in bleach pulp mill effluent [in Spanish]. Afinidad 1997;469:207 – 12. Vidal G, Videla S, Diez MC. Molecular weight distribution of Pinus radiata kraft mill wastewater treated by anaerobic digestion. Bioresour Technol 2001;77:183 – 91. Videla S, Diez MC. Experiences in wastewater treatment in Chilean forest industry. Water Sci Technol 1997;35:221 – 6. Ward NR, Wolfe RL, Justice CA, Olson BH. The identification of Gramnegative nonfermentative bacteria from water: problems and alternative approaches to identification. Adv Appl Microbiol 1986;31:294 – 7.
Aerobic degradation of 2,4,6-TCP content in ECF bleached effluent J. Correa a, V.M. Domı´nguez b, M. Martı´nez a, G. Vidal b,* a
Department of Microbiology, Biological Sciences Faculty, University of Concepcio´n, P.O. Box 160-C, Concepcion, Chile b Environmental Science Center EULA-Chile, University of Concepcio´n, P.O. Box 160-C, Concepcion, Chile Received 4 September 2002; accepted 12 December 2002
Abstract Elemental chlorine-free (ECF) bleach effluents from kraft mill are characterised by: a chemical organic demand/biological organic demand (COD/BOD5) ratio of 4, chlorophenol content with low chlorine substitution, and toxicity. The effect of increasing the concentration of 2,4,6-trichlorophenol (2,4,6-TCP) content in ECF bleaching sequence effluent on the degradative activity of bacterial communities present in an aerobic system treatment was studied. An aerobic lagoon (AL) was used as a typical secondary treatment of kraft-mill effluent. AL displays a high performance of BOD5 degradation (up to 90%); however, only 40% of the COD was removed. Simultaneously, the AL system shows a high ability to biodegrade 2,4,6-TCP up to 237 mg/l day. Kinetic parameters of the 2,4,6-TCP biodegradation by aerobic bacteria were determined. The Ks and Ki values were 34.3 and 50 mg/l 2,4,6-TCP, respectively. Moreover, the tolerance of aerobic bacteria was observed up to 1.3 g/l 2,4,6-TCP. D 2003 Elsevier Science Ltd. All rights reserved. Keywords: 2,4,6-TCP; Aerobic biodegradation; Isolation strain; ECF effluent
1. Introduction The pulp-and-paper industry has made substantial changes to pulping and bleaching technologies in order to minimise the formation and discharge of chlorinated organic material into aquatic environments (Videla and Diez, 1997). In the bleach plant, the major change has been the replacement of elemental chlorine dioxide, resulting in elemental chlorine-free (ECF) bleach effluents with lower absorbable organic halogens (AOX) and lower toxicity (Stauber et al., 1996). The biological demand for oxygen (BOD5) is reduced in the secondary treatment; however, specific compound removal (such as chlorophenols) is not always monitored. Diverse chlorolignin derivates of both low molecular weight (such as chlorophenols, chlorocatecols, chloroguaiacols) and high molecular weight are generated when ECF technologies are implemented (Lage et al., 1999; Vidal et al., 2001). In particular, 2,4,6-trichlorophenols (2,4,6-TCPs) are found as a major component of ECF bleach effluents (Andreoni et al., 1998; Diez et al., 2002). The study of the
* Corresponding author. Tel.: +56-41-204002; fax: +56-41-242546. E-mail address: [email protected] (G. Vidal).
role of microorganisms in chloroorganic removal during the aerobic treatment of bleached kraft-mill effluents (BKMEs) has been carried out recently (Fulhorpe et al., 1993). Studies show that chlorinated phenolics removal can be mineralized by aerobic bacteria in consortium (Vidal et al., 1997; Graves et al., 1995; Hall and Randle, 1994), or in a pure culture (Valenzuela et al., 1997; Domı´nguez et al., 2002). However, there are no specific studies that show evidence of the biodegradation of 2,4,6-TCP in effluents generated by the ECF process when Pinus radiata is used as the raw material. In order to predict the behaviour of these compounds, it is important to consider the kinetic biodegradation in the aerobic system. The Andrews kinetics (Eq. (1)) was used to describe the inhibition of 2,4,6-TCP and to evaluate the inhibition constant (Gu and Korus, 1995), considering the initial rate of the substrate degradation (rs) for each concentration evaluated (Vidal et al., 1997): rs ¼
dS X lm S ¼ dt YX =S Ks þ S þ SK2 i
ð1Þ
where S is the substrate concentration, X is the cell concentration, Ks is the Monod half saturation constant, Ki is an inhibition constant, lm is the maximum growth rate, and YX/S is the cell yield.
0160-4120/03/$ - see front matter D 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0160-4120(03)00002-3
460
J. Correa et al. / Environment International 29 (2003) 459–465
The aim of this work was to investigate the effect of increasing the concentration of 2,4,6-TCP content in ECF bleaching sequence effluent on the degradative activity of bacterial communities present in an aerobic treatment system.
2. Materials and methods 2.1. Wastewater The ECF effluent was obtained from a kraft mill described by Vidal et al. (2001). The effluent was supplemented with (NH4)2SO4 and KH2PO4 as a nitrogen and phosphate source (BOD5:N:P = 100:5:1). A stock solution of 2000 mg/l 2,4,6-TCP (Sigma, St. Louis, MO, USA) neutralized with NaOH solution (0.1 N) was prepared. Different 2,4,6-TCP concentrations were added to the ECF effluent. Table 1 shows the main physical – chemical characteristics of this effluent.
2.4. Kinetics of 2,4,6-TCP biodegradation The kinetics of 2,4,6-TCP biodegradation was measured using a batch system. Erlenmeyer flasks (250 ml) were incubated with biomass from the AL. Concentrations of 5, 10, 20, 40, and 80 mg/l 2,4,6-TCP were inoculated according to Domı´nguez et al. (2002). All of the assays were carried out in triplicate. The flasks were incubated in a shaker at 150 rpm, in the dark, at 25 F 2 jC. The variation of the 2,4,6-TCP concentration and cell concentration was analyzed according to the procedure described in Analytical Methods, during a period of 5 days, until total mineralization. The 2,4,6-TCP concentration was determined in the liquid phase for each assay by spectrophometry according to the process described by Aranda et al. (1999). The sample extraction was carried out according to the instructions of Veith and Kiwus (1997). The complete mineralization of the 2,4,6-TCP was confirmed by high-performance liquid chromatography (HPLC). 2.5. Isolation strains and bacterial viability
2.2. Inoculum A consortium of aerobic bacteria [4.1 g/l volatile suspended solid (VSS) and 5.9 g/l total suspended solid (TSS)] was inoculated in an aerobic lagoon (AL). This consortium arises from an activated sludge system that treats bleached kraft-mill effluent. 2.3. Continuous biodegradation system An AL (3.4 l) was used for biological effluent treatment. As is indicated in Fig. 1, an AL consists of an aerated zone and a settling zone. Oxygen concentration was maintained above 2 mg/l by a diffuser system. The AL operation was divided in two phases. In phase 1, the system was fed by means of a peristaltic pump with only ECF effluent; the hydraulic retention time (HRT) and organic load rate (OLR) in the system were 2 days and 184 mg/l day BOD5, respectively. In phase 2, the system was fed with ECF effluent supplemented with increasing 2,4,6-TCP concentrations; the OLR and HRT were 69 mg/l day BOD5 and 5 days, respectively. The toxic load rate (TLR) was maintained between 5.5 and 237 mg/l day 2,4,6-TCP.
Table 1 Physical – chemical characteristics of ECF effluent Parameter
Range
Averagea
pH COD (g/l) BOD5 (g/l) Total phenolic compounds UV215 (mg/l) Phenols (mg/l) Tannin and lignin (mg/l) TSS (g/l)
3.5 – 10.6 0.8 – 1.9 0.2 – 0.5 190.0 – 350.0 0.9 – 1.2 44.0 – 64.0 1.2 – 3.1
7.3 1.2 0.3 321.7 1.1 52.2 2.2
a
Average values correspond to 11 determinations.
Bacterial viability was determined in R2A agar plates and incubated at 25 jC for 5 days (Herbert, 1990; Godoy et al., 1999; Martı´nez et al., 1999). All experiments were carried out in triplicate. Samples obtained after 3 days of incubation were placed on R2A agar plates and incubated for 48 h at 25 jC in order to isolate bacteria strains that are present in ALs and that have the ability to degrade 2,4,6-TCP. Bacterial colonies with different morphological characteristics were first selected and frequently checked for purity in R2A agar plates and then characterised for Gram-strain type. Gramnegative bacilli were categorised by oxidative or fermentative capabilities in glucose H2S medium (Ward et al., 1986). The isolated cells were incubated in Erlenmeyer flasks (250 ml) at 25 jC with constant shaking (150 rpm) using the same method described by Godoy et al. (1999). Minimum medium (MM) was added to each of the seven Erlenmeyer flasks (containing 5, 10, 20, 40, 80, 160, and 360 mg/l 2,4,6TCP, respectively)—with the MM, effluent, and 2,4,6-TCP being the sole carbon and energy source. Additionally, we investigated the biodegradation of 2,4,6-TCP using R2A broth in order to evaluate the behavior of bacterial strain growth (Domı´nguez et al., 2002). The 2,4,6-TCP biodegradation and bacterial growth samples from each Erlenmeyer flask were analyzed daily for 7 days. 2.6. Analytical methods VSS, TSS, chemical organic demand (COD), biological organic demand (BOD5), phenols, and total phenolic compounds (UV215) were measured according to standard methods (APHA-AWWA-WPCF, 1985). Samples for determining the concentration of COD, BOD5, and UV215 were membrane-filtered (0.45 Am). The concentration of UV215
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Fig. 1. Aerobic lagoon: (1) influent, (2) pump, (3) diffusers, (4) aeration zone, (5) settling zone, (6) final effluent.
was determined in a 1-cm quartz cuvette by diluting the samples to less than 0.8 absorbance units (0.025 M borate was used as a buffer providing a pH of 9.1).
3. Results and discussion 3.1. Aerobic treatment Fig. 2 and Table 2 show the AL performance during 147 days of operation in which the TLR ranged from 5.5 to 237 mg/l day 2,4,6-TCP (Fig. 2a). The reactor was kept stable during the whole operation, maintaining the pH of the effluent between 7 and 8. In the first phase (0 –25 days of operation), when only ECF effluents were fed, almost 40% wt/wt of the COD was removed by the aerobic system (Fig. 2b). In contrast, BOD5 removal was between 90% and 96% wt/wt, whereas tannin and lignin removal was only around 13 –45% wt/wt. However, total phenolic compounds, measured as UV215, were poorly removed (8– 25% wt/wt), which indicates only a minor removal of these kinds of compounds. The influent COD/BOD5 ratio was 4, indicating that kraft-mill effluent is only partially biodegradable. Similar results were shown by Diez et al. (2002). Moreover, they found that the effluent biodegradation is highly influenced by the HRT in the activated sludge system; optimal HRT in this case was 48 h. In the second phase of the AL operation, ECF effluent was supplemented with increasing concentrations of 2,4,6TCP. The aerobic bacteria consortium displayed the ability to biodegrade the 2,4,6-TCP. However, during the operation between 26 and 36 days (TLR of 4 mg/l day 2,4,6-TCP), the aerobic system showed an accumulation of 2,4,6-TCP. Fig. 3a shows the profile of the 2,4,6-TCP content in the AL effluent; absorbance at 285 nm showed that the accumulation of the chlorophenols occurred between 26 and 34 days of operation, after which the 2,4,6-TCP was degraded. This phenomenon was also observed in each case when the TLR of 2,4,6-TCP was increased. Fig. 3b shows the 2,4,6-
TCP behaviour in the AL effluent, between 79 and 89 days of operation (TLR of 44 mg/l day 2,4,6-TCP). These results suggest that aerobic bacteria can degrade 2,4,6-TCP in the presence of ECF bleached effluent with high molecular weight (Vidal et al., 2001) and content of COD (mainly chloroaromatic containing), but need an adaptation period at a high concentration of the toxic. After that period, removal efficiency of 2,4,6-TCP during the process was always above 95%. Graves et al. (1995) also observed removal of chlorophenols from ECF effluent. They found AOX removal ranging from 15% to 43%, while removal of the individual chlorinate species varied from 12% to 100%. Hence, the concentration and viability of the microorganism population are important in the performance of the AL. In our experiments, the microorganism concentration increased from 2.38 to 3.37 g/l SSV and the ratio SSV/SST was between 0.76 and 0.68. Under these conditions, the 2,4,6TCP did not affect the metabolic activity of the microorganisms when the concentration of the toxic was increased from 20 to 1300 mg/l 2,4,6-TCP. Apart from the bacteria identifications in the system, we also observed the presence of a significant protozoan population in the AL, which reinforced the ability of the aerobic system to degrade the 2,4,6-TCP. 3.2. Kinetics of 2,4,6-TCP biodegradation In order to determine the kinetic parameters of the aerobic bacteria, we evaluated in batch system the bacterial capability to degrade 5, 20, 40, and 80 mg/l 2,4,6-TCP until mineralization. 2,4,6-TCP concentration was supplied with ECF effluents as the sole carbon and energy source in these assays. Assays with initial concentrations of 2,4,6-TCP between 20 (Fig. 4b) and 40 mg/l (Fig. 4c) showed a complete degradation of the chlorophenol at 120 h. After this, biomass could grow to 1.0 109 cfu/ml. The increase in biomass after the complete degradation of 2,4,6-TCP has been described as an intracellular dynamics that is often associated with phenol metabolism (Kim and Hao, 1999).
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Fig. 2. Performance of AL. (a) TLR (.). (b) COD (o) and 2,4,6-TCP removal (.). (c) Cell growth log (n).
A different behaviour was displayed in assays with 80 mg/l 2,4,6-TCP (Fig. 4d). A lag phase of about 50 h was observed. This indicates the toxic effect of the chloropheTable 2 Performance of aerobic lagoon during the operation Period (days)
Influent TLR (mg/l day 2,4,6-TCP)
2,4,6-TCP (mg/l)
2,4,6-TCP (% wt/wt)
0 – 25 26 – 55 56 – 75 76 – 86 89 – 107 110 – 133 134 – 147
0 5.5 12 44 72 114 237
0 30 70 250 400 650 1300
0 76.1 87.5 98.3 96.3 99.1 97
TLR = toxic load rate.
Effluent
nols and its inhibitory effect on the specific enzymatic activities in a batch system. Similar results were observed by Valenzuela et al. (1997). In our assays, no biodegradation was observed until 50 h. Also, from 24 to 50 h, the population increased to 1.0 104 cfu/ml. However, adaptation was observed and 80 mg/l 2,4,6-TCP was completely degraded at 120 h. The maximum degradation rate (1.1 mg/l h 2,4,6-TCP) and cell density (1.0 107 cfu/ml) were measured in the period higher than 120 h. The Andrews model was considered in order to obtain the main kinetic constant (saturation and inhibitory constant) of this aerobic bacteria in the presence of 2,4,6-TCP. Model parameters were determined by minimising the deviations between experimental data and model predictions of the degradation rate; the r2 value was 0.99. Thus, 34.3 and 50 mg/l were determined as saturation (Ks) and inhib-
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Fig. 5. Initial degradation rate of 2,4,6-TCP. Experimental data (n) and prediction by the Andrews model (—).
Fig. 3. Behaviour of 2,4,6-TCP in continuous system at TLR of (a) 5.5 mg/l day and (b) 44 mg/l day.
ition (Ki) constants, respectively, using Table Curve software. Fig. 5 shows the model fitted to the experimental data. The different model parameters for the degradation of 2,4,6-TCP (Table 3) can be explained as the difference in substrate transport across the membranes of the different cells. Little is known about how aromatic compounds and other hydrophobic compounds enter cells—although diffusion is the most cited mechanism (Reardon et al., 2000). Table 3 shows values of Ks and Ki similar to those obtained by Gu and Korus (1995) and Vidal et al. (1997).
Fig. 4. Degradation of 2,4,6-TCP with MM (.) and cell growth (o). Initial concentration of 2,4,6-TCP in each assay: (a) 5 mg/l, (b) 20 mg/l, (c) 40 mg/l, and (d) 80 mg/l.
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Table 3 Kinetic constants determined by the Andrews model in the degradation of 2,4,6-TCP Cells source Aerobic consortium from aerated lagoon-treated pulp kraft-mill effluent (raw material: pine) Isolated strain from river sediment Aerobic consortium from aerated lagoon-treated pulp kraft-mill effluent (raw material: eucalyptus) Flavobacterium sp. ATCC 39723 Consortium of six isolated and identified strains
Ks (mg/l)
Ki (mg/l)
Reference
34.3
50
This works
107 48.1
14 n.d.
156 n.d.
of COD (average 1.2 g/l COD) and toxic compounds. As a result, the bacteria needed to simultaneously develop both the ability to degrade nontoxic organic matter and to degrade toxic compounds. The study of the synergism phenomena between bacteria and the mechanism of the 2,4,6-TCP biodegradation in the presence of a co-substrate will be the objective for future works.
Domı´nguez et al. (2002) Vidal et al. (1997)
21
Gu and Korus (1995)
610
Kharoune et al. (2002)
n.d. = not determined.
Moreover, the values obtained in this work are very similar to those obtained by Vidal et al. (1997), who studied bacteria indigenous to Spain rather than Chile. However, in both cases, the substrates were ECF effluents and an aerobic consortium from AL-treated kraft-mill effluent. The value of the Ki constant (50 mg/l 2,4,6-TCP) helps to explain the performance of the continuous AL system. That means that aerobic biomass can be inhibited at concentrations below or similar to 50 mg/l 2,4,6-TCP; however, adaptation of the aerobic bacteria can improve the system performance. 3.3. Isolation strains Six strains were isolated in order to study the ability to degrade 2,4,6-TCP. Unfortunately, we did not observe evidence of the ability of the strains to degrade the toxic compounds. However, tolerance to 2,4,6-TCP was noted in an isolated condition, inoculated in R2A broth or saline solution (MM) with 2,4,6-TCP. Interestingly, degradative activity was detected only in the presence of cellulose effluent or in R2A broth inoculated with effluent (data not shown). Both conditions include different kinds of aerobic bacteria. The bacteria strains, in isolated condition, were unable to degrade 2,4,6-TCP when used as the sole carbon source or added to R2A broth, thus suggesting that degradative activity is a consequence of a bacterial consortium activity (Kharoune et al., 2002). Another possibility is that bacteria with degradative properties are found in viable but not cultivable conditions (Kejelleberg et al., 1987). In previous studies, we have isolated two different strains (Shingofixis chilensis S37, Ralstomia sp.) from river sediment with the ability to degrade 2,4,6-TCP (Domı´nguez et al., 2002; Godoy et al., 1999, 2002; Martı´nez et al., 1999). In both cases, strains were in contact with a low content of organic matter (below 10 mg/l COD). In contrast to these studies, we exposed aerobic bacteria to high concentrations
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